This research will be done primarily in Brazil at University Sao Paolo, in collaboration with Silvio and Monica Duailibi, as an extension of NIH grant # R01 DE016132-01. Our long-term goal is to define cell populations and methods that can be used for autologous tooth tissue engineering in humans. The ability to identify and manipulate adult tooth progenitor cells, or dental stem cells (DSCs), would significantly facilitate this process. We have previously demonstrated, in published reports, the successful use of both pig and rat tooth bud cells to generate bioengineered dental tissues. These results demonstrate our ability to isolate and maintain in culture, both epithelial and mesenchymal post-natal DSCs, and to use these cells to bioengineer small, accurately formed tooth crowns containing dentin, enamel, pulp, cementum, and periodontal ligament tissues. Based on these promising results, we hypothesize that human tooth bud cells can be used to generate bioengineered dental tissues. Here we propose studies whose successful completion will define methods to bioengineer human dental tissues. Specifically, we will: 1) Optimize harvesting/culturing conditions for human post-natal tooth bud cells; 2) Perform molecular/cellular characterization of human post-natal dental stem cells; and 3) Generate and characterize bioengineered human tooth tissues. The successful completion of the proposed aims will significantly improve our knowledge and understanding of human postnatal DSCs and their potential utility in tooth tissue engineering, and will provide the foundation for eventual autologous tooth tissue engineering in humans. [unreadable] [unreadable] [unreadable]